Cyanide Metabolism in Higher Plants: IV. Purification and Properties of The β-Cyanoalanine Synthase of Blue Lupine

Abstract

Abstract β-Cyanoalanine synthase, an enzyme which catalyzes the formation of β-cyanoalanine from l-cysteine and cyanide, has been isolated from the mitochondria of 10-day-old etiolated blue lupine seedlings. The enzyme can be assayed by measuring colorimetrically the sulfide that is also formed during the reaction. A partially purified fraction has been obtained with a specific activity that is 1700 times higher than that of an extract of whole seedling acetone powders. The purification procedure started with an extract of a mitochondrial acetone powder and involved the following steps: MnCl2 treatment, 0 to 80% (NH4)2SO4 precipitation, 38 to 52% (NH4)2SO4 precipitation, 50 to 75% acetone precipitation, Sephadex G-100 column chromatography, and preparative gel electrophoresis. An over-all yield of about 10% was achieved with an 140-fold increase in the specific activity over that of the initial extract of the mitochondrial acetone powder. β-Cyanoalanine synthase has a pH optimum of about 9.5 and a molecular weight of about 53,000 calculated from its elution volume on Sephadex G-100. The enzyme is stable and requires no added cofactors in the reaction mixture. The Km values for l-cysteine and cyanide are 2.5 mm and 0.55 mm, respectively, in the reaction forming β-cyanoalanine. The reverse reaction between β-cyanoalanine and sulfide does not occur under the conditions of the forward reaction. The enzyme is highly specific for l-cysteine with O-acetyl-l-serine being the only other substrate that will react with cyanide. However, the rate of β-cyanoalanine formation with O-acetyl-l-serine is only 5.4% of the rate observed with cysteine. The enzyme will also catalyze an exchange of sulfide into cysteine at one-half the rate of β-cyanoalanine formation. In addition, the purified enzyme will form cysteine from O-acetyl-l-serine and sulfide, but at only one-twenty-fifth of the rate of β-cyanoalanine formation. Methanethiol can replace cyanide as a substrate for the enzyme and in this case S-methylcysteine is formed. However the reaction rate is about one-sixth the rate of β-cyanoalanine formation with all substrates at 2.5 mm. During the purification it was also shown that O-acetylserine sulfhydrase, the enzyme that apparently synthesizes cysteine from O-acetylserine and sulfide in the blue lupine, is a soluble enzyme. This protein has an approximate molecular weight of 41,000 as judged from its elution volume on Sephadex G-100. O-Acetylserine sulfhydrase can also form β-cyanoalanine from O-acetylserine and cyanide; the rate for this reaction, however, is only one-tenth the rate of cysteine formation.